Enzymatic methods for genotyping on arrays

Abstract

The invention relates to methods for enzymatic, genotyping of polymorphisms on solid supports. In some aspects the methods include ligation of allele or base specific interrogation probes to an array probe. The array probe is labeled by ligation of the interrogation probe. Ligation is dependent on the identity of the base immediately adjacent to the end of the array probe. In other aspects array bound probes are labeled by template dependent extension.

What is claimed is: 
     
       1. A method for genotyping a plurality of target polymorphisms in a nucleic acid sample, the method comprising:
 obtaining an array comprising a plurality of array locations wherein each array location comprises a plurality of array probes of the same sequence covalently attached to a solid support, wherein the array probes in an array location are perfectly complementary to the sequence that is immediately 5′ of a target polymorphism in a target sequence, wherein said polymorphism is a single base change and wherein each array probe terminates at a base that is perfectly complementary to a first allele of said target polymorphism but not to a second allele of said target polymorphism, wherein said array also comprises a second array location comprising array probes that are perfectly complementary to the second allele; 
 hybridizing the nucleic acid sample to the array to allow array probes and target sequences to form complexes, wherein said complexes comprise a double stranded duplex region comprising a target sequence hybridized to an array probe and a single stranded region comprising target sequence, and washing to remove nucleic acid that is not hybridized specifically to an array location of the array; 
 adding a collection of labeled interrogation probes to the array to allow hybridization of the labeled interrogation probes to said single stranded region of said target sequences on the array, wherein said labeled interrogation probes are at least 6 bases in length and of random sequence; 
 adding a ligase to allow labeled interrogation probes to be ligated to the array probes in a template dependent manner; 
 washing the array to remove nucleic acids that are not covalently attached to the array; 
 detecting a hybridization pattern and determining from the hybridization pattern the presence or absence of labeled interrogation probe at array locations of the array and thereby determining the genotype of a plurality of target polymorphisms. 
 
     
     
       2. The method of  claim 1  wherein the plurality of polymorphisms comprises single nucleotide polymorphisms, single base deletions and single base insertions. 
     
     
       3. The method of  claim 1  wherein the labeled interrogation probes represent all possible sequences of 6 to 9 bases in length. 
     
     
       4. A method for genotyping a plurality of polymorphisms, wherein each of said plurality of polymorphisms is a single base change, the method comprising:
 obtaining an array comprising a plurality of array locations wherein each array location comprises a plurality of array probes of the same sequence covalently attached to a solid support, wherein the sequence is perfectly complementary to the sequence that is immediately 5′ of a target polymorphism and wherein each array probe terminates at a base immediately adjacent to a polymorphism; 
 hybridizing genomic DNA to be genotyped to the array wherein said genomic DNA hybridizes to complementary array probes to form a double stranded region and a single stranded 3′ region of the genomic DNA; 
 washing to remove genomic DNA that is not hybridized specifically to an array location of the array; 
 adding a collection of labeled interrogation probes to the array to allow hybridization of the labeled interrogation probes to the single stranded 3′ region of the genomic DNA wherein the labeled interrogation probes are differentially labeled depending on the base at the 3′ end of each labeled interrogation probe, are at least 6 bases in length, and are of random sequence; 
 adding a ligase to allow the labeled interrogation probes to be ligated to the array probes in a template dependent manner; 
 washing the array to remove nucleic acids that are not covalently attached to the array; 
 detecting a hybridization pattern; and 
 determining the genotype of a plurality of polymorphisms by determining the label or labels ligated to the array probes. 
 
     
     
       5. The method of  claim 3  wherein the labeled interrogation probes represent all possible sequences of 6 to 9 bases in length. 
     
     
       6. A method for determining the genotype of a plurality of polymorphisms, wherein each of said plurality of polymorphisms is a single base change, the method comprising:
 obtaining an array comprising at least 1,000,000 different target specific array probes wherein each target specific array probe is support bound at a different known or determinable location in the array, wherein each different target specific array probe is specific for one polymorphism in said plurality of polymorphisms and each different target specific array probe is specific for a different polymorphism in said plurality of polymorphisms; 
 hybridizing a target sample to the array to form complexes between the target specific array probes and complementary target sequences in the target sample, wherein said target sequences hybridize to complementary target specific array probes to form a double stranded region and a single stranded region of hybridized target sequence; 
 optionally removing unhybridized target sequences; 
 adding a plurality of labeled interrogation probes and a ligase to allow ligation of labeled interrogation probes to target specific array probes, wherein the labeled interrogation probes hybridize to the single stranded region of the hybridized target region immediately adjacent to the double stranded region, are at least 6 bases in length, are differentially labeled depending on the base at the 3′ end of each labeled interrogation probe, and are of random sequence; and 
 removing unligated labeled interrogation probes and determining the location of ligated labeled interrogation probes to determine the genotype of at least some of said plurality of polymorphisms. 
 
     
     
       7. The method of  claim 6  wherein the labeled interrogation probes are a mixture of 5′-NNNNNNNNN-3′ differentially labeled depending on the identity of the 3′ base. 
     
     
       8. The method of  claim 6  wherein the array comprises at least a first array probe and a second array probe for each polymorphism to be genotyped and wherein said first array probe and said second array probe are identical in sequence except at the base present at the 5′ end of the array probe. 
     
     
       9. The method of  claim 6  wherein for the array probes that are complementary to the target at a region that does not include the polymorphism but is immediately adjacent to the polymorphism, the labeled interrogation probe hybridizes to a region of the target that includes the polymorphism. 
     
     
       10. The method of  claim 6  wherein the labeled interrogation probes represent all possible sequences of 6 to 9 bases in length.